The plasma membrane of a cell separates the cytoplasm of the cell from the environment, and it is primarily composed of a phospholipid bilayer and proteins embedded within the bilayer or attached to the surface thereof. Normally the plasma membrane functions as a gate-keeper which allows essential substances to enter and exit the cell. However, the cell plasma membrane is a selective permeability barrier which blocks the passage of many useful therapeutic agents: hydrophilic molecules, highly charged molecules and macromolecules such as peptides and oligonucleotides, e.g., nucleic acid or gene, cannot be transported across the plasma membrane. Therefore, there has been a need for a reliable means of transporting drugs and macromolecules into cells.
Heretofore, a number of transporter molecules have been proposed to escort molecules across biological membranes. The proposed transporter molecules are lipids having positively charged residues, polymers of positively charged residues such as poly-lysine, and dendrimers having positively charged residues. However, such lipids, polymers and dendrimers have a common problem in that they are not easily soluble or biodegradable, and hence, precipitate in a cell to induce toxicity.
The basic region (i.e., AA 49-57) of Tat protein, which is a necessary trans-activator of HIV virus reproduction, has been reported to play a critical role in the process of the protein permeation through the plasma membrane. Proteins having a PTD (i.e., protein transduction domain) like the Tat basic region, that allows permeation through the plasma membrane include Antennapedia (Antp) homeodomain protein, Herpes virus protein VP22, Nuclear localization signal (NLS) sequence and the like, as shown in Table 1.
TABLE 1ProteinSEQhavingIDPTDAmino acid sequenceNO:HIV-1 TatGRKKRRQRRRPPQ1(48-60) AntpRQIKIWFQNRRMKWKK2(43-58) VP22DAATATRGRSAASRPTERDRAPARSASRPRRPVE3(267-300) SV40-NLSPKKKRKVC4 Nucleo-KRPAAIKKAGQAKKKKC5plasmin NF-kBPMLKQRKRQA6 HIV-1 RevRQARRNRRRRWRERQRG7(34-50) FHV CoatRRRNRTRRNRRRVRRGC8(35-49)
The above proteins seem to be capable of permeating across biological membranes without help of any specific receptor or transporter associated with the cell. Further, they share a common feature in that they mainly consist of basic amino acids, especially arginine and lysine.
Various hypotheses have been proposed with regard to the transmembrane mechanism of these proteins. One of the most plausible among them is that such proteins having a PTD can be transported into a cell by endocytosis-like process. Lebleu et al. (2003) determined using fluorescence activated cell sorter (FACS) that HIV-1 Tat (AA 48-60) and arginine nonamer (Arg9) are transported into a cell by endocytosis. However, the basic mechanism thereof has not yet been elucidated (Lebleu, B. et al., Biol. Chem., 278, 585, (2003)).
Further, there have been reported various studies to prepare oligomers having a plurality of arginine residues so as to have a high permeability into a cell. For example, Mann et al. (1991) showed that Tat protein is effective in enhancing the transportation of molecules attached thereto across a biological membrane. However, he also reported the problem that the total number of Tat proteins actually delivered into a cell is limited, because they are water-insoluble and too strongly bound to the cell surface, which leads to agglomeration among themselves (Mann, D. A. et al., EMBO J., 10, 1733 (1991)).
Barsoum et al. (1994) showed that shorter fragments of Tat protein (AA 1-72) and other fragments of the Tat protein containing the Tat basic region (AA 37-58) are also effective in enhancing the transportation of molecules attached thereto across a biological membrane. This study has shown that small peptides composed of amino acid residues contribute to the enhanced transportation of the molecules attached thereto across a biological membrane (Barsoum, J. et al., Proc. Natl. Acad. Sci. U.S.A., 91, 664(1994)).
Futaki et al. (2001) examined membrane permeabilities into mouse macrophage RAW264.7 cell of various peptides having a plurality of arginine residues by way of attaching thereto a fluorescent tag. The result revealed that a peptide having many arginine residues shows a permeability similar to that of Tat protein (AA 49-57), and an oligomer having eight (8) arginine residues is most effective in enhancing the transportation of molecules attached thereto across a biological membrane (Futaki, S. et al., J. Biol. Chem., 276, 5836 (2001)). These studies suggest that the guanidinium group of arginine is essential in the transportation of molecules attached thereto across a biological membrane.
Wender et al. (2000) designed a peptoid molecular transporter based on the fact that the biological membrane permeability depends on the number of the guanidinium group in a peptide, the length of the linker chain and chirality etc. Specifically, he noted that an L-arginine nonamer is 20-times more effective in the transportation across a biological membrane than Tat protein (AA 49-57), and a D-arginine nonamer is also significantly more effective in the transportation into a Jurkat cell, as was determined using FACS. Accordingly, the permeability of a peptoid having guanidinium groups is not significantly affected by the chirality of the amino acid (U.S. Pat. No. 6,495,663 and Wender, P. A. et al., Proc. Natl. Acad. Sci. U.S.A., 97, 13003 (2000)). However, such polyarginine peptide or peptoid molecules have the problems of rapid metabolism and elimination through the liver and kidney as well as their in vivo toxicity liability.
The present invention is based on the finding that inositol derivatives prepared from myo-inositol and a plurality of positively charged guanidinium groups significantly enhances the transportation of various therapeutic molecules attached thereto across a biological membrane.